Water-borne pathogens kill an estimated 1.7 million people annually and pose a serious threat to both national security in the United States and international economic development (Ashbolt N J. 2004. Microbial contamination of drinking water and disease outcomes in developing regions. Toxicology 198(1-3):229-38; Leclerc H, Schwartzbrod L, Dei-Cas E. 2002. Microbial agents associated with waterborne diseases. Crit Rev Microbiol 28(4):371-409). The enteric disease cholera affects developing nations throughout the world, especially in warmer climates such as Bangladesh (Guerrant R L, Carneiro-Filho B A, Dillingham R A. 2003. Cholera, diarrhea, and oral rehydration therapy: triumph and indictment. Clin Infect Dis 37(3):398-405). Caused by the marine bacterium Vibrio cholerae, the disease is marked by diarrhea and severe dehydration. A widely-considered low number for estimated deaths by cholera is between 120,000 and 200,000 deaths annually (Sanchez J, Holmgren J. 2005. Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea. Current Opinion in Immunology 17(4):388-398). Defense against this and other enteric diseases is hampered by their large scale, relative poverty of the outbreak areas, and lack of specificity in the treatment options: that is, when broadband antimicrobials are used to fight V. cholerae infection, it opens up the intestinal tract for colonization by opportunistic pathogens such as Clostridium difficile. 
The intestinal tract is home to at least 395 phylotypes of bacteria (Eckburg P B, Bik E M, Bernstein C N, Purdom E, Dethlefsen L, Sargent M, Gill S R, Nelson K E, Relman D A. 2005. Diversity of the human intestinal microbial flora. Science 308(5728):1635-8). These commensal bacteria (probiotics) have co-evolved with their host to provide nutrients, protect against pathogens, and aid in intestinal development (Holzapfel W H, Haberer P, Snel J, Schillinger U, Huis in't Veld J H. 1998. Overview of gut flora and probiotics. Int J Food Microbiol 41(2):85-101). Both pathogenic and non-pathogenic bacteria in the gut are known to use density-dependent cell to cell signaling (quorum sensing) to coordinate their growth and virulence (Kaper J B, Sperandio V. 2005. Bacterial cell-to-cell signaling in the gastrointestinal tract. Infect Immun 73(6):3197-209). For this reason quorum sensing has emerged as having tremendous potential for aiding in the control of pathogenic growth in the gut and elsewhere. Although there has been some success with using quorum sensing against pathogenic bacteria (March J C, Bentley W E. 2004. Quorum sensing and bacterial cross-talk in biotechnology. Curr Opin Biotechnol 15(5):495-502; Xavier K B, Bassler B L. 2005. Interference with AI-2-mediated bacterial cell-cell communication. Nature 437(7059):750-3), the full potential of this approach has been hampered by a lack of knowledge about the function of quorum sensing and about ways to exploit what knowledge exists. There have also been successful attempts to use commensal bacteria in preventing cholera disease symptoms through non-quorum-related mechanisms (Focareta A, Paton J C, Morona R, Cook J. Paton A W. 2006. A recombinant probiotic for treatment and prevention of cholera. Gastroenterology 130(6):1688-95). However, no one has yet to demonstrate the successful use of cell-to-cell signaling in preventing an invading pathogen from exhibiting virulence.
V. cholerae uses quorum sensing to coordinate its infection of the human GI tract (Miller M B, Skorupski K, Lenz D H, Taylor R K, Bassler B L. 2002. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell 110(3):303-14). When at a low cell density, V. cholerae expresses virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). TCP allows the invading V. cholerae to attach to the inside of the GI tract (Taylor R K, Miller V I, Furlong D B, Mekalanos J J. 1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci USA 84(9):2833-7) and CT triggers diarrhea and dehydration by stimulating adenylate cyclase (Moss J, Vaughan M. 1979. Activation of adenylate cyclase by choleragen. Annu Rev Biochem 48:581-600) (FIG. 1B). At higher cell densities, TCP and CT expression abates and expression of proteases that degrade the attachment matrix commences through a quorum-regulated circuit (Zhu J, Miller M B, Vance R E, Dziejman M, Bassler B L, Mekalanos J J. 2002. Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 99(5):3129-34).
While the purpose of this mechanism is not fully understood, it has been proposed that having virulence so timed allows for detachment and either relocation within or emergence from the human host once a high population density has been reached (Zhu J, Miller M B, Vance R E, Dziejman M, Bassler B L, Mekalanos J J. 2002. Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 99(5):3129-34) (FIG. 1A).
FIG. 1 shows a schematic of V. cholerae's infection cycle and quorum sensing circuit. At low cell density in the gut (FIG. 1A), V. cholerae (VC, ovals) expresses virulence factors cholera toxin (CT, pentagons) and toxin co-regulated pilus (TCP, strands), which infect the host epithelial cells (epithelia, rectangles) and allow VC to attach to the epithelia, respectively. At high cell density in the gut, VC stop expressing virulence genes and can therefore detach and leave the host with the efflux of fluid.
Two autoinducing molecules have been linked to quorum-related gene control in V. cholerae, cholera auto-inducer 1 (CAI-1) and auto-inducer 2 (AI-2). FIG. 1B shows the quorum network of V. cholerae: CqsA produces the autoinducer signal CAI-1 and LuxS produces the autoinducer signal AI-2. These systems converge with System 3 at Lux O to down-regulate virulence gene expression at high densities. High cell densities result in accumulation of CAI-1 and AI-2 to convert the signal cascade from kinase to phosphatase activity, repressing the transcription of sRNAs responsible for allowing virulence. (OM=outer membrane, IM=inner membrane).
There is a third component to the quorum regulatory circuit in V. cholerae (System 3), but this has been shown to act internally, without an external signal (Miller M B, Skorupski K, Lenz D H, Taylor R K, Bassler B L. 2002. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae (Cell 110(3):303-14)). CAI-1 is encoded by the gene cqsA in V. cholerae and AI-2 is encoded by the gene luxS.
V. cholerae El Tor serotypes are largely responsible for outbreaks of cholera in the developing world. The infection cycle for some strains of V. cholerae is coordinated, at least in part, through quorum sensing. That is, the expression of virulence genes depends on the concentration of V. cholerae autoinducers cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). High concentrations of CAI-1 and AI-2 have been shown previously to inhibit virulence gene expression.
There is therefore a need in the art for methods for using cell-to-cell signaling to prevent an invading pathogen from exhibiting virulence. There is also need in the art for recombinant microorganisms that are engineered to express signaling molecules that allow for communication with either the host's cells or with other bacteria either existing within or invading the host.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.